1. Field of the Invention
This invention relates to signal generating systems for specific binding assays. More particularly, it relates to improvements in the labeling of nucleic acid hydridization assay probes with nonradioactive moeities, such as biotin and other known labeling moieties.
2. Brief Description of the Prior Art
Oligonucleotides, including oligodeoxynucleotides, (oligomers) are now widely used as probes for the detection of specific genes and other nucleic acid sequences. The most common method for the labeling of oligonucleotides has been the incorporation of the isotope .sup.32 P. While this approach is suitable for research purposes, the safety precautions required, together with the relatively short half life of .sup.32 P have stimulated the search for effective, non-radioactive labeling methods.
Among the variety of non-radioactive labels thus far reported, the vitamin biotin (and biotin analogues such as iminobiotin) has attracted considerable attention due to its high affinity binding with avidin and streptavidin, versatility, and ease of handling, in addition to the sensitivity of the detection systems in which it is used. Biotinylated probes have been used for the sensitive colorimetric detection of target nucleic acid sequences on nitrocellulose filters, as shown in Leary, et. al., PNAS 80:4045(1983), and for in situ detection of target DNAs, as in Langer-Safer, et. al., PNAS, 79:4381(1982).
The enzymatic incorporation of biotin into DNA is well documented; incorporation of a biotinylated nucleoside triphosphate can be accomplished by nick translation procedures, as discussed in Langer, et. al., PNAS 78:6633(1981), or by terminal addition with terminal deoxynucleotide transferase, as described by Brakel, et. al., in Kingsbury, et. al. (Eds.), Rapid Detection and Identification of Infectious Agents, Academic Press, New York, pgs. 235-243(1985) and in Riley, et. al., DNA, 5:333(1986). Biotin labels have also been introduced into DNA using a photochemical method. See, for example, Forster, et. al., Nuc. Ac. Res., 13:745(1985).
Several groups have concentrated on the development of methods for the attachment of a single biotin at the 5'-end of an oligonucleotide, either via a phosphoramidate, as in Chollet, et. al., Nuc. Ac. Res., 13:1529(1985) and in Wachter, et. al., Nuc. Ac. Res., 14:7985(1986), or a phosphodiester linkage. Regarding the latter, see Kempe, et. al., Nuc. Ac. Res., 13:45(1985); Agrawal, et. al., Nuc. Ac. Res., 14:6227(1986); and Chu, et. al., DNA, 4:327(1985). Although these methods can be used to provide oligonucleotide probes, only one biotin group per oligomer is introduced by these methods.
In order to increase the sensitivity of biotinylated oligonucleotide probes, efforts have been directed to the introduction of several biotins in such a way that the hybridization of the oligomer is not impaired. This has been accomplished by enzymatic methods, using terminal transferase, as in Riley, et. al., supra, or Klenow fragment, as in Murasugi, et. al., DNA, 3:269(1984). Also, chemical procedures for the introduction of multiple biotins on sidearms attached to internal nucleotides of a probe have been described in a preliminary report by Bryan, et. al., DNA, 3:124(1984).